Growth hormone (GH), also referred to as somatotropin, plays an important role in animal growth and development. It regulates a variety of physiological effects, including linear growth of the animal, lactation, differentiation and electrolyte balance. The molecular mechanism of these biological effects involves the binding of gowth hormone to a specific plasma membrane receptor, referred to as growth hormone receptor (GHR).
Growth hormones from different species share a significant level of sequence homology. Human GH (huGH) is a polypeptide chain of 190 amino acids (aa) and a molecular weight of 22 kDa, while rat GH is 189aa long and has 64% sequence homology to its human counterpart. Growth hormone binds to a GHR, which consists of three domains: an extracellular hormone-binding domain, which is 28 kDa for the human GHR, a single pass transmembrane domain and an intracellular domain, which is 35 ka for the human GHR. A soluble form of the extracellular domain occurs naturally in blood as a growth hormone binding protein (GHBP). The molecular interactions between GH and membrane-bound GHR are thought to be analogous to those between GH and soluble GHBP. Receptor activation requires simultaneous binding of two GHR by one GH, i.e., receptor dimerization, to form a complex wherein the two intracellular domains can initiate the process of signal transduction underlying GH activity.
Rat GHBP (rGHBP), like its membrane bound form (rGHR), is fully cross-reactive with both human and rat GH (rGH). The human GHR, and human GHBP, can bind only the human GH (huGH) and not the rGH.
The ability to control the activation of GHR is important in developing new therapies for certain diseases such as dwarfism and acromegaly. Recombinant huGH is presently on the market as a drug for dwarfism. Injectable formulations of bovine GH also are used in animal husbandry to promote growth and milk production in cows.
Although proteins have become highly visible as potential drugs, their use as therapeutics presents several difficult problems, including the high cost of production and formulation, administration via injection and limited stability in the bloodstream. Therefore, replacing proteins, including GH, with a small molecular weight drug has received a lot of attention. However, none of these efforts has resulted in finding a successful drug. A key problem to replacing GH, as with some other proteins, is that a small molecule that binds to one receptor site would only be an antagonist. A dimer of two small molecules, capable of binding to two receptor units and dimerizing them, is needed for agonist activity. Up to now there is no assay for such dimers.
The present invention is focused on finding peptides that specifically recognize the sites involved in activation of proteins of pharmacological importance. Once identified and characterized as regulators of target activity, these peptides may be used in high throughput screens to identify and provide information on small molecules which bind at these sites and, when dimerized, mimic the function of GH.
Yanofsky et al. (PNAS 93:7391-7386) describes the isolation of a monomer peptide antagonistic to IL-1 with nanomolar affinity for the IL-1 receptor. This effort required construction and use of many phage-display peptide libraries and sophisticated phage panning procedures.
Wrighton et al. (Science, 278:458-463) and Livnah et al. (Science, 273: 469-471) reported dimer peptides that bind to the erythropoietin (EPO) receptor with fall agonistic activity in vivo. These peptides are cyclical and have intra-peptide disulfide bonds; like the IL-1 receptor antagonist, they show no significant sequence identity to the natural ligand. Importantly, X-ray crystallography revealed that it was the spontaneous formation of non-covalent homodimers that enabled it to dimerize two EPO receptors.
Most recently Cwirla et al. (Science, 276:1696-1699) described the identification of two families of peptides that bind to the human thrombopoietin (TPO) receptor and are competed by the binding of the natural ligand TPO. The one with highest affinity, when dimerized by chemical means proved to be as potent an in vivo agonist as TPO.
The present invention provides assays for identifying compounds that mimic the binding characteristics of growth hormone. Such compounds would serve as antagonists of growth hormone function. Dimers of such compounds would serve as growth hormone agonists.
The present invention also provides polypeptides that inhibit binding of growth hormone to growth hormone receptor. Such polypeptides are used in the assays of the invention to identify compounds that mimic growth hormone.